SUMMARY A large variety of chemicals require evaluation for organ-specific toxicity. Animals have traditionally been used for toxicological screening; however this mode of evaluation is expensive, ethically challenged and not always predictable. In vitro monolayer has been used as an alternative, however cells in this format lose the specialized interactions requisite for differentiation and optimal function. Thus, 3D in vitro organotypic cellular models (OCM) that recapitulate key in vivo features of are being sought. The kidney poses a particularly difficult challenge due to its highly complex spatial and functional orientation that has not been replicated in vitro. Emphasis is now being placed on creation of in vitro assays that mimic specific structural micro- environments. We have fabricated a 3D in vitro kidney medullary organotypic culture model (MOCM) utilizing mouse ureteric bud (UB) and metanephric mesenchymal (MM) progenitor cells in co-culture in sea sponge scaffolds that mimic the natural tubular/vessel micro-environment. The cells in this system reciprocally induce each other to form organoids comprised of collecting duct epithelium surrounded by capillary profiles. The objective of this grant is to validate that the MOCM platform can be used for in vitro screening of toxins that affect medullary development and mature cell systems. Aim 1 will validate the 3D MOCM platform as an in vitro prototype for assessment of toxic agents on medullary development. The UB and MM cells produce the growth factors and possess their cognate receptors for reciprocal induction of collecting duct epithelium and vasculogenesis. Toxic effects of known inhibitors of nephrogenesis including imatinib mesylate (an inhibitor of platelet-derived growth factor receptor (PDGFR-?), NVP-AST-487 (an inhibitor of glial cell-derived neurotrophic factor (GDNF) receptor RET, and captopril (an inhibitor of angiotensin converting enzyme (ACE) will be evaluated for toxicity of MOCM development. Aim 2 will validate the 3D MOCM platform as in vitro prototype for the assessment of toxic agents on established (mature) medullary structures. Our UB cells express AQP2 and are responsive to vasopressin. Lithium is a known pharmaco-chemical toxin of collecting duct principal cells via interference with vasopressin regulation of a water channel protein aquaporin 2 (AQP2). Mature MOCM scaffolds will be treated with vasopressin to enhance AQP2 and the effect of lithium toxicity on this expression will be evaluated. Cell toxicity in both aims will be evaluated using assessments of organoid structure and immunoreactivity of AQP2 and other marker proteins using immunohistochemistry, immunoblotting and ELISA. Data will be compared to the effects of these agents on human and rodent medullary structures in vivo as reported in the literature. This MOCM system has the potential to provide an innovative platform to facilitate toxicant effects on the kidney medulla and will reduce animal testing.